Antibacterial agents comprising conjugates of glycopeptides and peptidic membrane associating elements

ABSTRACT

The invention concerns agents with anti-bacterial activity and methods and intermediates for their production. The present invention further concerns the use of such agents for the treatment of bacterial infections in animals, including man. The agents are derivatives of vancomycin-type antibiotics, of structure: V-L-W—X; wherein V is a glycopeptide moiety which inhibits peptidoglycan biosynthesis in bacteria; L is a linking group; W is a peptidic membrane-associating element such as an element based on naturally-occurring animal or bacterial peptide antibiotics; and X is hydrogen or a membrane-insertive element.

[0001] The present invention concerns agents with anti-bacterialactivity and methods and intermediates for their production. The presentinvention further concerns the use of such agents for the treatment ofbacterial infections in animals, including man.

BACKGROUND OF THE INVENTION

[0002] Diseases caused by bacterial infections have significantmorbidity and mortality in man and other mammals. The infection processconsists of three stages: bacterial entry and colonization of the Host;bacterial invasion and growth in host tissues along with the appearanceof toxic substances; and the host response.

[0003] Bacterial infections can be classed broadly into those caused byGram positive bacteria, such as the Staphylococci and Streptococci, andthose caused by Gram negative bacteria, such as Escherichia coli. Grampositive bacteria have a typical lipid bilayer cytoplasmic membranesurrounded by a rigid cell wall. The cell wall is composed mainly ofpeptidoglycan, a polymer of N-acetylglucosamine and N-acetyl muramicacid crosslinked by a peptide comprising alternating D- and L-aminoacids. In addition, the outer cell wall of Gram-positive bacteriacomprises a complex of polysaccharides, proteins, teichoic acids, andlipoteichoic acids. By contrast, Gram-negative bacteria have a muchsmaller peptidoglycan layer, an outer membrane that containslipopolysaccharide which lacks the complex layer of carbohydrate andteichoic acids.

[0004] Antibiotics are substances produced by various species ofmicroorganisms (bacteria, fungi) that suppress the growth of othermicroorganisms and may eventually destroy them. In addition, commonusage extends the term antibiotic to include antibacterial agents whichare semi-synthetic antibiotics, i.e. chemically modified bacterialantibiotics, as well as synthetic antibacterial agents (e.g.sulphonamides) which are not products of microbes. Also included in theterm “antibiotic” are various peptides found in host defence systemswhich are produced locally in response to colonisation by or invasion ofmicroorganisms (e.g. peptides produced by amphibians, including thepeptide magainin). Hundreds of antibiotics have been identified, andmany have been developed to the stage where they are of value in thetherapy of infectious diseases.

[0005] Several schemes have been proposed to classify and groupantimicrobial agents. The most common classification has been based onchemical structure and proposed mechanism of action, as follows: (1)agents that act directly on the cell membrane of the microorganism,affecting permeability and leading to leakage of the intracellularcompounds, such as detergents, cationic peptides, gramicidin A, andpplymyxin; (2) agents that inhibit synthesis of bacterial cell walls andincludes the beta-lactams, cephalasporins and glycopeptides; (3) agentsthat affect bacterial protein synthesis including tetracycline andchloramphenicol; (4) agents that act as antimetabolites and interferewith the bacterial synthesis of folic acid, such as the sulphonamides;and (5) agents that inhibit nucleic acid synthesis or activity such asquinolones.

[0006] Peptide anti-bacterial agents which act directly on the bacterialmembrane cause a general permeabilisation or modification of thebacterial cytoplasmic membrane. This results from the binding ofpeptides to components of the outer membrane surface, causingreorganisation of membrane structure and the creation of pores throughwhich the intracellular contents may leak. Generally, these features areassociated with an amphiphilic peptide nature often including helicalsecondary structure and a net positive charge. Peptide antibioticshaving this mode of action include the magainins, defensins, and thelantibiotics such as nisin. The activity of, this class of antibioticsis directed towards bacteria rather than mammalian cells because thepositive charged residues of the antibiotic interact with negativelycharged lipids which are found predominantly in bacterial rather thanmammalian cell membranes.

[0007] In particular, the magainins are a class of amphiphilic α-helicalpeptides found in the skin of the African clawed frog (Xenopus laevis).Peptides of this class (which also include bombinin from amphibians(Gibson, B. W., Tang, D., Mandrell, R., Kelly, M. & Spindel, E. R.(1991) J. Biol. Chem. 266, 223103-23111], melittin from bee venom[Habermann, E. (1972) Science, 177, 314-322], and alamethicin from fungi[Latorre, R. & Alvarez, S. (1981) Physiol. Rev., 61, 77-150]) causedisruption of membrane potential at low concentrations, and membranelysis via insertion at higher concentrations.

[0008] One group of antibiotics that has received widespread attentiondue to their clinical efficacy is the glycopeptide group of antibiotics.These agents consist of a rigid, cyclised heptapeptide backbone whichmay be substituted with a variety of amino and non-amino sugars. Theamino sugar moieties of some members of this class contain N-acyl,N-alkyl, or N-aryl substitutions. Two antibiotics in this class arevancomycin and teicoplanin. Vancomycin is produced by Streptococcusorientalis, an actinomycete isolated from soil samples in Indonesia andIndia. The antibiotic was purified and its properties described shortlyafter its discovery (McCormick et al., 1956). Vancomycin is a complextricyclic glycopeptide with a molecular mass of approximately 1500 Da.Its structure was determined by X-ray analysis (Sheldrick et al., 1978):

[0009] Vancomycin is active primarily against Gram-positive bacteria.Strains of bacteria are considered susceptible at a minimum inhibitoryconcentration of less than or equal to 4 μg/mL. Strep. Pyogenes, Strep.Pneumoniae, Corynebacteraium spp. are highly susceptible, as are moststrains of Enterococcus spp. Most species of Actinomyces and Clostridiumspp. are also sensitive to vancomycin, but at higher concentrations ofantibiotic. Vancomycin is employed only to treat serious infections andis particularly useful in the management of infections due tomethicillin-resistant staphylococci, including pneumonia, emphysema,endocarditis, osteomyelitis, and soft-tissue abscesses. The agent isalso extremely useful in the treatment of staphylococcal infections inpatients who are allergic to penicillins and cephalosporins.

[0010] Vancomycin inhibits the synthesis of the cell wall in sensitivebacteria by blocking the cross-linking of the sugar and peptidiccomponents of peptidoglycans during the synthesis of the bacterial cellwall. Without sufficient cross-linking, the cell wall becomesmechanically fragile and the bacteria lyse when subjected to changes inosmotic pressure. Vancomycin binds with high affinity to theD-alanyl-D-alanine (D-Ala-D-Ala) terminus of the pentapeptide portion ofthe peptidoglycan precursor before cross-linking. The D-Ala-D-Aladipeptide forms complementary hydrogen bonds with the peptide backboneof vancomycin. It is thought that the vancomycin-peptidoglycan complexphysically blocks the action of the transpeptidase enzyme and therebyinhibits the formation of the peptide cross-bridges that strengthens thepeptidoglycan. This activity also leads to the accumulation ofpeptidoglycan precursors in the bacterial cytoplasm.

[0011] Resistance to antibiotics is well documented and the resistantstrains are a potential major threat to the wellbeing of mankind.Bacteria become resistant to an antimicrobial agent because either thedrug fails to reach its target; the drug is inactivated, or because thetarget is altered. For example, some bacteria produce enzymes thatreside in or within the cell surface and inactivate the drug, whileothers possess impermeable cell membranes that prevent influx of thedrug.

[0012] Several types of resistance have been described for vancomycin,including the VanA-C types. The VanA phenotype is inducible byvancomycin and confers resistance to both teicoplanin and vancomycin.The VanA phenotype is mediated by the transposable element Tn1546 orclosely related elements (Arthur et al., 1993). The transposon encodes adehydrogenase (VanH) that reduces pyruvate to D-lactate (D-lac), and aligase of broad substrate specificity (VanA) that catalyses theformation of an ester bond between D-Ala and D-Lac (Dukta-Malen et al.,1990; Bugg et al., 1991). The resulting D-Ala-D-Lac depsipeptidereplaces the dipeptide D-Ala-D-Ala in the pathway of peptidoglycansynthesis. The substitution eliminates a hydrogen bond that is criticalfor antibiotic binding (Bugg et al., 1991). The VanB phenotype is alsoinduced upon exposure to vancomycin; however, in contrast to the VanAphenotype, these microorganisms are not resistant to teicoplanin becauseteicoplanin does not induce the expression of the genes required forresistance in VanB bacteria (Arthur et al., 1996; Evers and Courvalin,1996). Resistance to vancomycin by bacteria of the VanB phenotype occursthrough a similar mechanism to VanA resistance, namely the substitutionof the terminal D-Ala-A-Ala peptidoglycan precursor on the immaturepeptidoglycan by the D-Ala-D-Lac depsipeptide. One furthervancomycin-resistant phenotype has been described (VanC) in enterococcibelonging to the species E. gallinarum, E. casseliflavus and E.flavescens. These bacteria are intrinsically resistant to low levels ofvancomycin and are susceptible to teicoplanin. Resistance results fromthe production of peptidoglycan precursors ending in D-Serine(Billot-Klein et al., 1994; Reynolds et al., 1994).

[0013] Substitution of D-Ala by D-Ser at the carboxy-terminus of thepeptidoglycan precursor analogues lowers the affinity of the precursorsfor vancomycin with a relatively small change in the affinity forteicoplanin (Billot-Klein et al., 1994). The emergence and disseminationof high-level resistance to glycopeptides in enterococci in the pastdecade has resulted in clinical isolates resistant to all antibiotics ofproven efficacy (Handwerger et al., 1992; Handwerger et al., 1993). Theincidence of glycopeptide resistance among clinical isolates isincreasing and enterococci have become important as nosocomial pathogensand as a reservoir of resistance genes (Murray 1990; Woodford et al.,1995). Nosocomial infection with multidrug resistant strains ispotentially catastrophic and there is a need to identify novelanti-bacterial agents or methods of controlling bacterial infections.

[0014] Approaches that have been used to combat the emergence ofantibiotic resistant strains include the modification of existingantibiotics to improve their potency against resistant organisms, or thediscovery of new peptide antibiotics which kill their targets bypermeabilizing the bacterial plasma membrane. Examples of the firstapproach have recently focussed on creating derivatives of glycopeptidessuch as vancomycin.

[0015] Functionalisation of the carboxyl terminal of vancomycin usingthe coupling agent2-(1-hydroxybenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) has been successful in attaching shortpeptide sequences, both in solution and solid phases [Sundram, U. N. andGriffin, J. H. (1995) J. Org. Chem. 60 1102-1103]. The aminosugar andterminal amine moieties of vancomycin and related antibiotics. have alsobeen derivatised. In a reductive alkylation approach, a series ofcompounds alkylated on the vancosamine sugar was created, some of whichshowed greatly improved activity vs vancomycin resistant bacterialstrains [Cooper, R. D. G. et al. (1996) J. Antibiotics 49, 575-581;Rodriguez, M. J. et al. (1998) J. Antibiotics 51, 560-569].

[0016] WO-A-98/02454 describes polypeptide derivatives in which asoluble therapeutic polypeptide is modified with an entity of generalstructure:

-(L-[W])n-X   (I)

[0017] in which each L is independently a flexible linker group, each Wis independently a peptidic membrane-binding element, n is an integergreater than or equal to one, and X is a peptidic or non-peptidicmembrane-binding or insertive element.

[0018] Structures of type (I) represent a combinatorial array ofmembrane-interactive elements whose attachment to soluble polypeptideswas found to mediate binding of those polypeptides to the outer cellmembrane of mammalian cells. This gave rise to therapeutic benefits,particularly in the case of regulators of complement activation actingas cytoprotectants and anti-inflammatory agents (e.g. Dong, J. et al,(1999) Mol. Immunol. 36 957-963).

SUMMARY OF THE INVENTION

[0019] The present inventors hypothesised that structures of type (I)would display even stronger binding to bacterial membranes which have ahigher proportion of acidic phospholipids than do eukaryotic organisms,and have a higher proportion of membrane-associated biosyntheticproteins, and it has now been found that the anti-bacterial activity ofcompounds such as vancomycin and its derivatives can be increased whenthey are derivatised further with structures of type (I), and relatedstructures.

[0020] Accordingly, a first aspect of the present invention provides acompound:

V-L-W—X   (II)

[0021] wherein

[0022] V is a glycopeptide moiety which inhibits peptidoglycanbiosynthesis in bacteria;

[0023] L is a linking group;

[0024] W is a peptidic membrane-associating element; and

[0025] X is hydrogen or a membrane-insertive element.

[0026] Peptidoglycan biosynthesis inhibitor (V).

[0027] The first two stages of peptidoglycan occur inside the bacterialcell. Stage 1 involves the assembly of a N-acetylmuramic acid basedlipid with a linked pentapeptide, the peptide being:

[0028] L-Alanine-γ-D-Glutamate-Xaa-D-Alanine-D-Alanine (SEQ ID NO:39),where Xaa is usually m-D-amino pimelic acid but in some species (e.g. S.aureus) is L-lysine.

[0029] In the second stage, the lipid is extended by N-acetylglucosamine. This lipid is subsequently transported across the cellmembrane.

[0030] The third stage, which takes place on the exterior surface of thebacterial membrane, involves the polymerization of the lipid-linkedGlcNAc-MurNAC-disaccharide by a transglycolase and the cross-linking ofthe peptide side chains by a transpeptidase.

[0031] The best known compound of the class of inhibitors of thisbiosynthesis pathway is vancomycin, which, as discussed above, is knownto inhibit peptidoglycan biosynthesis by binding to the D-Ala-D-Aladipeptide terminus of the pentapeptide of the bacterial cell wallpeptidoglycan precursors, preventing their further processing intopeptidoglycan.

[0032] Derivatives of vancomycin also act by inhibiting the biosynthesisof peptidoglycan. A series of compounds alkylated on the vancosaminesugar has been shown to have activity against vancomycin resistantbacteria, along with analogous compounds derivatized with a furthersugar [Cooper, R. D. G. et al. (1996) J. Antibiotics 49, 575-581;Rodriguez, M. J. et al. (1998) J. Antibiotics 51, 560-569; Ge, M. et al.(1999) Science 284, 507-511].

[0033] In general, the moiety V is a glycopeptide moiety which inhibitspeptidoglycan biosynthesis in bacteria. In general terms, those of skillin the art are familiar with glycopeptides of this class and may selectsuitable glycopeptides for use in the present invention. Suchglycopeptides are typically of a molecular weight of from 1000 to 3000Da, are capable of interaction with individual components of thebacterial peptidoglycan structure such as the Lys-D-Ala-D-Ala peptide,the Lys-D-Ala-D-Lactate depsipeptide, and components of the lipidGlcNAc-MurNAC-pentapeptide, and are active againstvancomycin-susceptible reference strains (e.g. selected from any one ofreference strains S. aureus NCTC (National Collection of Type Cultures)6571, S. aureus ATCC 25923 (NCTC 12981), S. aureus ATCC 29213 (NCTC12973), Streptococcus pneumoniae ATCC 49619 (NCTC 12977) andEnterococcus faecalis ATCC 29212 (NCTC 12697)) at a mic of less than orequal to 4 μg/ml. An accepted standard method for mic testing is theagar dilution method on IsoSensitest agar as recommended by the BSAC. Itis published in The Journal of Antimicrobial Chemotherapy (1991), vol.27, supplement D.

[0034] Particular vancomycin derivatives which are contemplated as themoiety V include compounds based on the glycopeptides disclosed in WO96/30401 and WO 98/00153, and salts thereof, the disclosures of whichare herein incorporated by reference.

[0035] Thus, preferred examples of the moiety V-L- include those offormula (III):

[0036] or salt thereof, in which:

[0037] Y and Y′ are independently hydrogen or chloro;

[0038] R is hydrogen, 4-epi-vancosaminyl, actinosaminyl, ristosaminyl,or a group of the formula —Ra-L- wherein Ra is 4-epi-vancosaminyl,actinosaminyl, ristosaminyl and L (the linker of formula (II)) isattached to the amino group of Ra;

[0039] R1 is hydrogen, or mannose;

[0040] R2 is —NH2, —NHCH3, —N(CH3)2, —NHL-, or —N(CH3)L-

[0041] R3 is —CH2CH(CH3)2, [p-OH, m-Cl]phenyl, p-rhamnose-phenyl,[p-rhamnose-galactose]phenyl, [p-galactose-galactose]phenyl, or[p-CH3O-rhamnose]phenyl;

[0042] R4 is —CH2—(CO)NH2, benzyl, [p-OH]phenyl, or [p-OH, m-Cl]phenyl;

[0043] R5 is hydrogen, or mannose,

[0044] R6 is hydrogen, 4-epi-vancosaminyl, vancosaminyl, actinosaminyl,ristosaminyl, or acosaminyl; or R6 is a group of the formula Rb-L-wherein Rb is 4-epi-vancosaminyl, vancosaminyl, actinosaminyl,ristosaminyl or acosaminyl and L is attached to the amino group of Rb;or R6 is a group Rb—R7 wherein R7 is an organic side chain moiety whichis no more than 1000, preferably no more than 500 and preferably no morethan 250 (such as no more than 150) Da;

[0045] Ter1 is hydroxy or -L-;

[0046] provided that the moiety includes at least one (for example twoor three) group(s) -L-.

[0047] The precise nature of the organic side chain moiety is not alimiting feature of the present invention. Many thousands of suchmoieties are known as such in the art, including the numerous examplesdescribed in WO 96/30401 and WO 98/00153, the disclosures of which areincorporated herein by reference.

[0048] A subgroup of organic side chain moieties include those of theformula —CH2-R8, in which R8 is:

[0049] hydrogen,

[0050] alkyl of C1-C15,

[0051] alkenyl of C2-C15,

[0052] alkynyl of C2-C15,

[0053] haloalkyl of C1-C7,

[0054] acenaphthenyl,

[0055] 2-fluorenyl,

[0056] 9, 10-dihydro-2-phenanthrenyl,

[0057] R9,

[0058] alkyl of C1-C11-R9,

[0059] alkenyl of C2-C7-R9,

[0060] alkynyl of C2-C7-R9, or

[0061] alkyl of C1-C7-O—R9

[0062] wherein R9 is a radical of the formula:

—R10-[linker(0 or 1)-R10](0 or 1)

[0063] wherein each R10 independently represents phenyl, cycloalkyl ofC5-C6, naphthyl, or thienyl, each of which is unsubstituted or isoptionally substituted with on or two substituents, each of which isindependently alkyl of C1-C10, haloalkyl of C1-C2, haloalkoxy of C1-C2,alkoxy of C1-C10, halo, cyano, or nitro;

[0064] and “linker” is:

[0065] alkylene of C1-C3,

[0066] —O-alkylene of C1-C6,

[0067] -alkylene of C1-C6,

[0068] —O—,

[0069] —N(H or lower alkyl of C1-C3)-,

[0070] —S—,

[0071] —SO—,

[0072] —SO2-,

[0073] —NH—C(O)—,

[0074] —C(O)—NH—

[0075] —CH═CH—,

[0076] —CC—,

[0077] —N═N—,

[0078] —O—C(O)—, or

[0079] —C(O)—O—

[0080] “Halo” means fluoro, chloro, bromo or iodo; fluoro and chloro arepreferred. Haloalkyl and haloalkoxy groups are preferablymono-substituted or di-substituted with the same halo group, althoughC1-3 haloalkyl groups may also be perfluoro groups.

[0081] Preferred examples of the group —CH2-R8 include:

[0082] (4-phenylbenzyl)

[0083] (4-(4-chlorophenyl)benzyl)

[0084] (4-(4-methylphenyl)benzyl)

[0085] (4-phenoxybenzyl)

[0086] ((4-n-butylphenyl)benzyl)

[0087] (4-benzylbenzyl)

[0088] Compounds of the formula (III) include LY 264826, LY 191145 andLY 333328 as disclosed in Rodriguez, M. J. et al. (1998) J. Antibiotics51, 560-569.

[0089] Particularly preferred examples of the moiety V include, but arenot limited to, vancomycin, chloroeremomycin, teicoplanin A2-2,ristocetin A, eremomycin, balkimycin, actinodin A, complestanin,chloropeptin 1, kistamycin A and avoparcin.

[0090] Peptidic membrane-associating element (W).

[0091] This moiety (W) of the compound of formula II is a peptide whichassociates-with the bacterial membrane. While not wishing to be bound byany one particular theory, it is believed that the association of theelement W with the bacterial membrane allows an increase in the localconcentration of the glycopeptide antibiotic such that its reducedefficiency against resistant strains is alleviated by the increasedconcentration at the site of action.

[0092] The term “membrane associating” refers to two primary modes ofaction, binding to and/or insertion in the membrane. In the case of theformer, the peptide is of a character which allows it to associate withelements on the surface of the bacterial membrane, such as negativelycharged phospholipids. In the case of the latter, the element may be, orbased upon, anti-bacterial peptides which have this property (forexample derived from those found in nature).

[0093] Peptides may be prepared recombinantly or synthetically, e.g. bystep-wise synthesis. Alternatively, the peptides may be recovered fromcultures of cells which naturally produce the peptide, e.g. in the caseof membrane associating peptides produced by bacteria.

[0094] Peptides produced by synthetic means will generally be composedof natural L-amino acids (i.e. those encoded by the genetic code, theso-called proteinogenic amino acids), although D-amino acids may also beused. In either case, side chain modifications may be performed, forexample in order to enhance in vivo half life or improve stability. Sidechain modifications include for example, modifications of amino groupsby reductive alkylation by reaction with an aldehyde followed byreduction with NaBH4, amidination with methylacetimidate or acylationwith acetic anhydride.

[0095] The guanidino groups of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione or glyoxal. Sulphydryl groups may be modified by methodssuch as carboxymethylation, tryptophan residues may be modified byoxidation or alkylation of the indole ring and the imidazole ring ofhistidine residues may be modified by alkylation.

[0096] The carboxy terminus and any other carboxy side chains may beblocked in the form of an ester group, e.g. a C1-6alkyl ester or in theform of an amide.

[0097] The N-terminus may also be blocked.

[0098] The above examples of modifications to amino acids are notexhaustive. Those of skill in the art may modify amino acid side chainswhere desired using chemistry known per se in the art.

[0099] Peptides recovered from naturally occurring sources may containnon-proteinogenic amino acids, which are produced either by posttranslational modification of proteinogenic amino acids, or bybiosynthesis.

[0100] The peptidic element may terminate with a cysteine or lysineresidue or have such a residue within 1 or 2 amino acids from theC-terminal, in order to facilitate linking to the group V via the linkerL. However, other amino acids are not excluded and may be used where thenature of the linking group is suitable for attachment to othermoieties.

[0101] The peptide element W is generally of a size from 5 to 40 aminoacids in length, preferably from 7 to 30 such as 8 to 30 amino acids.

[0102] It will be understood that unless indicated to the contrary,amino acid sequences are represented herein using standard notation andin the N- to C-terminal direction.

[0103] Membrane-binding peptides.

[0104] Where the peptide is membrane binding, the element (W) maycomprise a number of charged amino acid residues generally selected fromarginine and lysine, particularly lysine, in order to facilitateinteraction with the charged lipids found in bacterial membranes. Suchpeptides preferably include a least one sequence (Xaa)n, where n is from1 to 12, preferably from 3 to 10, and each Xaa is independently lysineor arginine.

[0105] Thus, taking account of the overall preferences referred toabove, W may be a peptide of from 5 to 40 amino acids comprising atleast one sequence of from 1 to 12, more preferably from 2 to 10 such asfrom 3 to 10 contiguous residues selected from lysine and arginine.

[0106] Even more preferably, W may be a peptide of from 7, preferably 8to 30 amino acids comprising at least one sequence of from 1 to 12, morepreferably from 2, preferably 3 to 10 contiguous residues selected fromlysine and arginine.

[0107] More preferably, W may be a peptide of from 7, preferably 8 to 30amino acids comprising at least one sequence of from 1 to 12, morepreferably from 2, preferably 3 to 10 contiguous lysine residues.

[0108] In all of the above embodiments, it is preferred that the peptidehas an overall positive charge, e.g. from +1 to +10. Examples of suchelements include:

[0109] DGPKKKKKKSPSKSSG (SEQ ID NO:4);

[0110] GSSKSPSKKKKKKPGD (SEQ ID NO:5);

[0111] SPSNETPKKKKKRFSFKKSG (SEQ ID NO:6);

[0112] DGPKKKKKKSPSKSSK (SEQ ID NO:7); and

[0113] SKDGKKKKKKSKTK (SEQ ID NO:8).

[0114] Membrane-inserting peptides.

[0115] Where the peptide is a membrane inserting peptide, such a peptideis one which itself has anti-bacterial activity, due to the action ofthe peptide disrupting the membrane, often by forming pores therein. By“antibacterial activity”, it is meant that the conjugate of the peptidelinked to vancomycin at the carboxy terminal of vancomycin, has an micof no more than 0.064 mg/ml, preferably no more than 0.032 mg/ml,against the E. faecalis strains referred to above under the conditionsreferred to above.

[0116] Particular examples of such peptides include those derived from anatural source, such as from an animal. Certain peptides derived from avariety of sources, such as amphibian skin, are known topossess-membrane inserting properties, often accompanied byantibacterial properties.

[0117] A large number of groups of such peptides are known to possesssuch activity, including peptides reviewed in Jack & Jung, Chimia 52(1988); 48-55, and McCafferty et al, Current Opinion in Chemical Biology(1999) 3; 672-680, the disclosures of which are incorporated herein byreference.

[0118] One group of peptides are α- and -defensins, produced by a widevariety of animal sources. The alpha defensins generally sharesubstantial degrees of homology, contain a large number of Arg residues(but not Lys) and the disulfide-bond arrangement is uniformly conserved;disruption of the disulfide bonds results in loss of antimicrobialactivity. The defensins are produced as prepro-peptides, and the leader-and pro-segments are probably involved in directing their transport tostorage vesicles to await use. The -defensin TAP was isolated frombovine mucosa; -defensins are larger than the α-defensins and have adifferent disulfide arrangement. A large number of different defensinpeptides and peptide families differing in their Arg/Lys content, numberand arrangement of disulfide bonds and of differing overall length havebeen isolated, including HNP-1 and the tachyplesins which are18-amino-acid antimicrobial peptides isolated from the horseshoe crab.The peptide TAP is part of a family of tachyplesins whose primarysequence varies slightly, but whose disulfide-bond arrangement andsecondary structure remains conserved.

[0119] A great number of defensin-like peptides have now been described,having been isolated from a variety of sources including immune cells ofmammals, mucosa, insect haemolymph, crustacea and plants and theirseeds.

[0120] A further group of antimicrobial peptides have also been isolatedfrom bacteria. Bacteriocins, or bacterial-derived antibacterialpeptides, are produced by many different bacterial species. Thestructures vary considerably: some contain disulfide bonds, some containfree cysteine, some contain neither cysteine nor cystine and a fourthgroup consists of two peptides whose complementary presence is requiredfor antimicrobial activity. However, regardless of their structuralcharacteristics, they all act by forming hydrophilic pores in thecytoplasmic membrane of susceptible bacteria. These pores or channelsdepolarise the cytoplasmic membrane, disrupting energy transduction andATP production.

[0121] One well-characterised bacteriocin is pediocin PA-1, produced byPediococcus acidilactici PAC1.0. The bacteriocin contains nopost-translational modifications, but does contain two essentialdisulfide bonds and a N-terminal sequence which is homologous with anumber of other bacteriocins.

[0122] Lantibiotics are also bacteriocins and therefore they areribosomally synthesised as precursor peptides. However, unlike thebacteriocins described above, the lantibiotics contain a large number ofposttranslational modifications; the name lantibiotic is derived fromtheir content in the thioether amino acids lanthionine and3-methyllanthionine.

[0123] More than 25 lantibiotics have been characterised. Mostlantibiotics are highly modified, many containing 50% (or more)non-proteinogenic α-amino acids.

[0124] In addition to the thioether amino acids, the lantibioticscontain a variety of other modified amino acids including2,3-didehydroalanine and -butyrine, lysino-alanine,2-aminovinyl-D-cysteine, hydroxy-aspartic acid, a number of N-terminalmodifications and D-Ala. Not all lantibiotics contain each of thesemodified amino acids; in some cases, they are widespread, whilst inothers they are specific to only one lantibiotic.

[0125] Particularly useful lantibiotics include type A lantibioticswhich form pores in energised membrane bilayers, resulting innonspecific, transient channels in the cytoplasmic membrane. Type Aantibiotics include nisin and galligermin, the sequences of which areillustrated in Jack & Jung, 1998, ibid.

[0126] The following peptides are examples of those which may be ofparticular use in the present invention: magainin 1 and 2(Xenopuslaevis) whose sequences are GIGKFLHSAGKFGKAFVGEIMK (SEQ ID NO:9) andGIGKFLHSAKKFGKAFVGEIMNS (SEQ ID NO:10) respectively, as well as those ofTable 1 below. TABLE 1 SEQ. Peptide Structure ID NO: GramicidinVGALAVVVWLWLWLW 11 Caerin 1.1 GLLSVLGSVAKHVLPHVVPVIAEHL 12 RanalexinFLGGLIKIVPAMICAVTKKC 13 Maculatin 1.1 GLFGVLAKVAAHVVPAIAEHF 14 GS14K4VKLKVYPLKVKLYP 15 Indolicidin ILPWKWPWWPWRR 16 polymyxin B cyclizedisooctanoyl 17 BTBB(BFdLBBT) CP26 KWKSFIKKLTSAAKKVVTTAKPLISS 18 CEMAKWKLFKKIGIGAVLKVLTTGLPALTLTK 19 CP29 KWKSFIKKLTTAVKKVLTTGLPALIS 20CP11-NH2 ILKKWPWWPWRRK-NH2 21 CEME KWKLFKKIGIGAVLKVLTTGLPALIS 22bactenecin Cyclized RL(CRIVVIRVC)R 23 linear Bac RLCRIVVIRVCR 24 Bac2SRLSRIVVIRVSR 25 gramicidin S cyclic (PFdLOVPFdLOV) 26 Gram4112 cyclic(PVKLKVdYdPLKVKLYd) 27 indolicidin ILPWKWPWWPWRR-NH2 28 MelittinGIGAVLKVLTTGLPALISWIKRKRQQ 29 [D]- GIGAdVLKdVLTTGLPALdISWIdKRKRQQ 30 V5,8I17K21 Melittin Pexiganan GIGKFLKKAKKFGKAFVKILKK 31

[0127] These and the other membrane inserting peptides referred toherein, or variants thereof such as those which have from 1 to 5 aminoacid substitutions, insertions or deletions and which retain theactivity defined above form a further group of the element W which maybe used.

[0128] Examples of such variants include:

[0129] GIGKFLHSAKKFGKAFVAEIMNS (SEQ ID NO:32);

[0130] GIAKFLHSAKKFGKAFVAEIMNS (SEQ ID NO:33);

[0131] AAGKFLHSAKKFGKAFVGDIMNS (SEQ ID NO:34);

[0132] G-GKFLHSAKKFGKAFVGEIMNS (SEQ ID NO:35);

[0133] G-GKFIHSAKKFGKAFVGEIMNS (SEQ ID NO:36);

[0134] GIGKPIHSAKKFGKAFVGEIMNSK (SEQ ID NO:37); and

[0135] GIGAVLKVLTTGLPALISWIKRKRQQC (SEQ ID NO:38),

[0136] where letters in bold show substitutions of, or additions to, thewild-type magainin or melittin sequences described above, and −indicates a deletion.

[0137] It is preferred that the peptides have an overall positivecharge, e.g. from +1 to +10.

[0138] Linking group L.

[0139] This linking group is all the atoms between the moiety V and themoiety W, and therefore the linking group must have at one end a moietycapable of linking to the peptidoglycan biosynthesis inhibitor (V), andat the other end a moiety capable of linking to the peptidicmembrane-binding element (W).

[0140] It will be appreciated by those of skill in the art thatcompounds of the invention are generally synthesised by reacting areactive derivative of W with a reactive derivative of V (with the groupX either being attached to W before or after this step). Thus thestructure -L- in the compounds of the invention will comprise atomswhich were part of the reactive derivatives of both V and W.

[0141] Methods of providing reactive derivatives of glycopeptides andpolypeptides are known per se in the art and those of skill in the artwill be able to select from a range of methodologies in order to link Vwith W, resulting in a group L. Thus while the exact nature of the groupL is not an essential feature of the invention, in one aspect this groupis conveniently represented as:

-A-R—B—  (III)

[0142] where A is a group capable of linking to the peptidoglycanbiosynthesis inhibitor (V), B is a group capable of linking to thepeptidic membrane-binding element (W), and R is a bond or a grouplinking A and B.

[0143] Examples of linking groups of this type are the chemical bridginggroups, for example as described in EP-A-109653, EP-A-152736,EP-A-155388 and EP-A-284413, the disclosures of which are incorporatedherein by reference.

Linking to W

[0144] If W is to be joined through its N-terminus, or via an aminemoiety on a side chain residue (e.g. the ε-amino group of lysine), thenB may be the radical of a moiety capable of reaction with an aminegroup. For example, the precursor to B may be a carboxylic acid (orderivative thereof), which when reacted with the N-terminus of theprecursor to W, results in B being —C(═O)—, linked to W with an amidebond. In these embodiments, it is envisaged that the link is formed byreacting B═—R═ with W═, where W═ is the precursor to W, B═ the precursorto B and R═ a precursor of the remainder of the compound of formula II.

[0145] Alternatively, B may be a N-acetyl radical, —C(═O)—CH2-T-, wherethe carboxyl carbon is attached to the amine group of W, and T isselected from O, S, NH. Such a linkage can be formed by synthesising theN-haloacetyl derivative of W, followed by reaction with a appropriateprecursor B′—R═, where B′ is either OH, SH, or NH2.

[0146] If W is to be joined through its C-terminus, then B may be theradical of a moiety capable of reaction with a carboxylic acid group,which is usually a nucleophile. For example, the precursor to B may bean amine (or derivative thereof), which when reacted with the C-terminusof the precursor to W, results in B being —NH—, linked to W with anamide bond. In these embodiments, it is envisaged that the link isformed by reacting B═—R═ with W═, where W═ is the precursor to W, B═ theprecursor to B and R═ a precursor of the remainder of the compound offormula II.

[0147] In the case where W terminates with a cysteine amino acid, thenthe linking group preferably terminates with sulphur, such that L joinsto W by disulfide or thioether linkage. Thus, in formula (III), B is—S—. This linkage may be formed by activating the S on either theprecursor to W or the precursor to B, for example, by forming a2-pyridyl disulphide derivative which can react with a thio group toform the desired disulphide link.

Linking to V

[0148] Where V is vancomycin, the point of attachment of the linkinggroup (L) is derived either from the amino terminus (2), from the amineof the vancosamine sugar (3) or, more preferably, from the carboxylterminus (1).

[0149] The means of derivatisation and linkage are as described above.

[0150] If V is a vancomycin derivative in which one of the abovepositions is already derivatised, then the point of attachment may be onone of the remaining available positions, or any suitable position onthe derivatisation.

[0151] In the case where the group R is not a bond, but a group linkingA and B, then it is preferably an alkylene chain containing from 3 to 12carbon atoms, which chain may be interrupted by one or more hetero-atomsand/or aromatic rings, e.g. benzene or pyridine, and may contain one ormore carbon-carbon double or triple bonds, and may be substituted withone or more functional groups.

[0152] Thus, R may include moieties which interact with water tomaintain the water solubility of the linking group and suitable moietiesinclude —CO—NH—, —CO—NMe-, —S—S—, —CH(OH)—, —SO2-, —CO2-, —(CH2CH2O)m-and —CH(COOH)—, where m is an integer of 2 or more.

[0153] Therefore examples of R include —(CH2)r-, —(CH2)p-S—S—(CH2)q- and—(CH2)p=—CH(OH)—CH(OH)—(CH2)q=-, in which r is an integer from 3 to 12,and p and q are independently integers whose total is from 3 to 12, andp= and q= are integers whose total is from 1 to 10.

[0154] Membrane-insertive element X.

[0155] This element is optionally present on compounds of the invention.Where W is a membrane binding peptide, the presence of a membraneinsertive element is preferred. Where W has these properties, thepresence of a further insertive element is not excluded.

[0156] A range of elements with membrane insertive properties are knownin the art. A preferred class which is contemplated by the presentinvention is a lipophilic chain based on carbon atoms. Many such chainsare known in the art and the precise nature of the primary chemicalstructure is not essential to the invention, provided that the elementis capable of having sufficient lipophilicity to partition intobacterial membranes when brought into contact with such membranes in anaqueous environment.

[0157] In general, the lipophilic chain based on carbon atoms is definedas:

[0158] having from 6 to 30, preferably from 6 to 24 carbon atomsincluding those of any aromatic rings, if present;

[0159] being straight or branched, and in the case of the lattercontaining one or more, for example two or three branch points;

[0160] being saturated or unsaturated, in the case of the lattercontaining one or more, for example 2, 3 or 4, double or triple bonds;

[0161] having 1, 2 or 3 heteroatoms (in addition to those, if present,in aromatic rings, if present, independently selected from S, O or N;

[0162] optionally containing one or more, for example two or three,aromatic rings, which may be fused and each of which may contain from 1,2 or 3 heteroatoms which, if present, are independently selected from N,O or S; and

[0163] optionally having one or more (such as 1, 2 or 3) substituentsselected from hydroxy, —SH, amino and halo (where halo is fluoro,chloro, bromo or iodo).

[0164] Preferably, the aromatic rings are six-membered, and may beselected from benzene and pyridine. If the rings are fused, then theymay be selected from naphthalene, anthracene, quinolene andisoquinolene. Other examples of aromatic rings include thiophene andpyrrole.

[0165] In one embodiment, the element comprises an uninterruptedaliphatic carbon chain of at least six atoms, with preferably no morethan 12, 16 or 20 carbon atoms. One group of such compounds have thestructure -Ph-O—(CH2)t-H, where Ph is a benzene ring and t is from 6 to12, 16, or 20.

[0166] In another, the element is a fatty acid, preferably those with 5to 28 carbon atoms, optionally containing up to four, more preferablyone or two carbon-carbon double bonds. Unsaturated radicals have thestructure —C(O)—(CH2)t=-H, where t= is from 5 to 27, more preferably 9,11, 13, 15, 17 or 19.

[0167] Another embodiment provides an element comprising the grouping—C(O)-Ph-Ph, where Ph is a phenyl group, which may be substituted, forexample to provide an element —C(O)-Ph-Ph-p-Cl.

[0168] Another embodiment is an ether group within a fatty acidcontaining from 10 to 16 carbon atoms in total, which optionallycontains one or two double bonds.

[0169] Another embodiment is a group of the formulaPh—CH(OH)—CH2—NH—C(Me2), where Ph is a phenylene ring substituted at thepara position by a group RO—CH2-and the meta position by a group RO—,where each R is independently a C4-10 alkyl chain.

Administration of Drug

[0170] A second aspect of the present invention is a pharmaceuticalcomposition comprising a compound of formula (II) and a pharmaceuticallyacceptable carrier.

[0171] The formulations optionally comprise other therapeuticingredients, or diluents. The carrier or carriers must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipients thereof.

[0172] Formulations suitable for parenteral or intramuscularadministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample water, for injections, immediately prior to use. Injectionsolutions and suspensions may be prepared extemporaneously from sterilepowders, granules and tablets.

[0173] It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations may include other agentsconventional in the art having regard to the type of formulation inquestion. Of the possible formulations, sterile pyrogen-free aqueous andnon-aqueous solutions are preferred.

[0174] Alternatively the composition may be formulated for topicalapplication for example in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures, and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions. Such carriers may constitute from about 1% to about 98% byweight of the formulation; more usually they will constitute up to about80% by weight of the formulation.

[0175] The composition of the invention may be administered by injectionto achieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections.

[0176] Many orthopaedic surgeons consider that patients with prostheticjoints should be considered for antibiotic prophylaxis before dentaltreatment that could produce a bacteraemia. Late deep infection is aserious complication sometimes leading to loss of the prosthetic jointand is accompanied by significant morbidity and mortality. It istherefore possible to extend the use of the peptide or peptide/drugconjugate as a replacement for prophylactic antibiotics in thissituation.

[0177] Bacterial infections cause one of the major complicationsassociated with the clinical use of implanted materials and in-dwellingdevices. In particular, staphylococci have frequently been implicated inmedical device-related infections (Dankert et al 1986, CRC RevBiocompatability 2, 219-301). Once established, the infection isvirtually impossible to treat resulting in implant failure. Attempts tocombat staphylococcal adhesion to implants have involved modification ofthe surface of the prosthetic material to discourage adhesion ofproteins; e.g. coating with a “non-stick” material such as PTFE, orbonding antibiotics to the surface (Kamal et al., 1991, J. Amer. Med.Assoc. 265, 2364-2368 ). In addition, there have also been proposals touse non-steroidal anti-inflammatory drugs to prevent adhesion ofstaphylococci to medical polymers (Farber and Wolff 1992, J. Infect.Dis. 166: 861-865).

[0178] For administration to human patients, it is expected that thedaily dosage level of the active agent will be from 0.01 to 50 mg/kg,typically around 1 mg/kg. The physician in any event will determine theactual dosage most suitable for an individual patient, and will varywith the age, weight, and response of the particular patient. The abovedosages are exemplary of the average case. There can, of course, beindividual instances where higher or lower dosage ranges are merited,and such are within the scope of this invention.

[0179] In addition to the therapy described above, the compositions ofthis invention may be used generally as a wound treatment agent toprevent adhesion of bacteria to matrix proteins, especially fibronectin,exposed in wound tissue and for prophylactic use in dental treatment asan alternative to, or in conjunction with, antibiotic prophylaxis.

[0180] Alternatively, the composition of the invention may be used tobathe an indwelling device immediately before insertion. The activeagent will preferably be present at a concentration of 0.1 g/ml to 10mg/ml for bathing of wounds or indwelling devices.

[0181] Compositions of the invention may be used for, but are notrestricted to, the treatment of bacterial infections caused by thefollowing organisms: Mycobacterium sp.; Enterococcus sp.; Escherichiasp.; Staphylococcus sp.; Streptococcus sp.; Vibrio sp.; Neisseria Sp.;Borrelia sp.; Klebsiella sp.; Hemophilus sp.; Clostridium sp.;Pseudomonas sp.; Actinomyces sp.; Pneumococcus sp.; Salmonella sp.

[0182] In a further aspects of the present invention, compounds offormula (II) may be used as a pharmaceutical or in methods of treatmentof the animal or human body, and in particular for treatment ofbacterial infections caused by the above listed organisms. Compounds offormula (II) may also be used in the manufacture of a medicament for thetreatment of bacterial infections, particularly those caused by theabove listed organisms.

EXAMPLES

[0183] Embodiments of the present invention will now be described indetail by way of example.

Methods Haemolysis Assay

[0184] Lysis of sensitised sheep erythrocytes was measured using astandard haemolytic assay using a v-bottom microtitre plate format asfollows:

[0185] 50 microlitres of a range of concentrations of compound dilutedin Hepes buffer were mixed with 100 microlitres of sensitised sheeperythrocytes and then incubated for 1 hour at 37° C. Samples were spunat 1600 rpm at ambient temperature for 3 minutes before transferring 150microlitres of supernatant to a flat bottom microtitre plate anddetermining the absorption at 405 or 410 nm. Maximum lysis (Amax) wasdetermined by incubating serum with erythrbcytes in the presence ofhuman serum diluted 1:400 (final concentration in assay mixture) in 0.1M Hepes/0.15 M NaCl/0.1% gelatin pH 7.4. Background lysis (Ao) wasdetermined by incubating erythrocytes in the absence of any serum orcompound, using PBS as a control. Lysis was expressed as a fraction of100% total cell lysis such that LC50 represents the concentration ofcompound required to give 50% lysis. Antibiotics with low lytic activityin erythrocytes compared to their antibacterial activity areadvantageous.

Antimicrobial Activity Assay

[0186] Compounds were tested for antimicrobial activity against avariety of bacterial strains that included one or more of the followingmicroorganisms: Escherichia coli strain TG1, Bacillus subtilis strain168S, Staphylococcus aureus H, Enterococcus faecium STR 207 (Van Aresistant phenotype), Enrterococcus faecium STR211 (Vancomycin sensitivephenotype), and Enterococcus faecalis V 583 (Van B resistant phenotype).For the testing of compounds for antimicrobial activity against E. coliand B. subtilis cultures of each bacterial strain were diluted in 2.5 mLof fresh LB broth to approximately 5×106 cells/mL for assay. Compoundsto be tested were diluted in water and added to the bacterial culturesto give final concentrations between 200 μg/mL and 0.5 ng/mL. Thecultures were grown with shaking for up to 16 h at 37° C. for E. colicultures and 30° C. for B. subtilis cultures. Antibacterial activity wasdetermined from inspection of the turbidity of the different culturesand from determination of the optical density of the cultures at awavelength of 600 nm. For the testing of compounds for antimicrobialactivity against E. Faecium STR 207, E. faecium 211, E. faecalis V 583,and S. aureus H, a different procedure was used. These microorganismswere cultured in brain heart/0.5% yeast extract (BHY) broth andincubated overnight at 37° C. A sample of each culture was then diluted40-fold in fresh BHY broth and incubated at 37° C. for 1 h. Theresultant mid-log phase cultures were diluted to 106 cfu/mL, then addedto wells of 96-well polypropylene plates. Vancomycin,was seriallydiluted 2-fold across the culture containing wells from 128 to 0.0156μg/mL. Test compounds were then serially diluted in a similar manneracross the culture containing wells from 512 μg/mL to 0.03 μg/mL. The96-well plates were covered and incubated at 37° C. overnight. Minimuminhibitory concentrations were determined by inspection of turbidityafter incubation.

Example 1 Synthesis and Characterisation of APT542

[0187] (MSWP-1, Example 2 of WO/9802454). The peptide:Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-Cys-NH2(SEQ ID NO:1) was prepared using solid phase synthesis via the generalFmoc/tBu strategy developed by Sheppard and Atherton (E. Atherton and R.C. Sheppard, Solid Phase Synthesis, IRL Press, Oxford, 1989).Kieselguhr-supported polydimethylacrylamide resin (Macrosorb 100) wasused as the solid support and was derivatised with ethylene diamine.

[0188] Coupling reactions were carried out using N-α-Fmoc protectedreagents pre-activated withN,N′-diisopropylcarbodiimide/N-hydroxybenzotriazole (in 4-fold molarexcess) with bromophenol blue monitoring. Fmoc Cleavages used 20%piperidine in DMF. Reactions to assemble the peptide chain were carriedout by repeated cycles of coupling and deprotection including theattachment of the modified Rink linkage reagent(p-[(R,S)-α-(1-(9H-fluoreny-9-yl-methoxyformamido]-2,4dimethoxybenzyl]-phenoxyacetic acid) designed to yield a C-terminalamide on final cleavage. The side chain functionalities of theindividual amino-acids were protected as follows: Ser (tButyl), Lys(Boc), Asp (O-tButyl), Cys (Trityl).

[0189] On completion of the peptide assembly and with the peptide stillattached to the resin, a myristoyl group was attached to the amino groupof the N terminal glycine by direct coupling of myristic acid using thesame activation procedure. This modified peptide was then cleaved fromthe resin and the side-chain protecting groups removed at the same timeby treatment with trifluoracetic acid containing 2.5% water and 2.5%triisopropyl silane.

[0190] The crude product was treated with 2,2′-dithiopyridine in 0.01 Mammonium acetate solution at pH 8-9 for approx. 2 h, then acidified withacetic acid and purified by preparative high performance liquidchromatography (HPLC) with 0.1% trifluoracetic acid (TFA)/water and 0.1%TFA/acetonitrile as gradient components. After lyophilisation, thepeptide was a white amorphous powder, soluble to at least 10 mg/ml indimethylsulphoxide. Fast atom bombardment mass spectrometry gave mainpeaks at m/e 2107.8, 2129.7 and 2145.8, corresponding to themonoprotonated, monosodiated and monopotassiated molecular ions of thepeptide. The 2-thiopyridyl content of the peptide was measured bydissolving it to around 0.03 mM to 0.2 mM in 0.1 M Sodium Borate pH 8.0and reducing by addition of dithiothreitol to 5 mM. The change inoptical density at 343 nm was used to calculate the amount of pyridine2-thione released using an extinction coefficient at this wavelength of8080 cm-l M-1. This indicated that the peptide content was approximately60% of the dry weight.

[0191] The final produce thus has thestructure:N-(Myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-2-thiopyridyl)-Cys-NH2APT542 was tested for antimicrobial activity against E. coli strain TG1and B. subtilis 168S as described in Methods. The minimum inhibitoryconcentration of APT542 to prevent the growth of E. coli strain TG1 was0.022 mg/mL after 6 h and 0.067 mg/mL after 16 h. The minimum inhibitoryconcentration of APT542 to prevent the growth of B. subtilis strain 168Swas 0.003 mg/mL after 6 h and 0.022 mg/mL after 24 h growth. Theantibacterial activity of APT542 was also determined against E. faeciumSTR 207, E. faecalis V 583, E. faecium STR 211 and S. aureus H, asdescribed in Methods. The minimum inhibitory concentration of APT542 was0.064 mg/mL, 0.256 mg/mL, 0.032 mg/mL, and 0.064 mg/mL for eachmicroorganism respectively.

[0192] Example 2

Synthesis and Characterisation of APT540

[0193] APT540 is a dimer of APT542, linked via a cysteine bridge throughthe C-terminal cys residues.

[0194] APT542 (Example 1; Example 2 in WO 98/02454; 54 mg) was dissolvedin 0.1 M Tris pH 8.5 (2.68 mL) and treated with 0.35 molar equivalentsof tris-2-carboxyethyl phosphine (TCEP) dissolved in water. The reactionwas allowed to proceed for 30 minutes at room temperature and analysedby HPLC using a C18 reverse phase column with a gradient of 35%-90%acetonitrile in 0.1% trifluoroacetic acid. Reaction products weremonitored at wavelengths of 210 nm and 310 nm. APT540 was identified asa peak eluting from the column at approximately 10.5 minutes afterinjection. The material corresponding to the APT540 compound wascollected and lyophilised. Mass spectrometry of this sample using aPerSeptive Biosystems instrument identified a major peak of 3998 daltonswhich corresponds to the theoretical molecular weight of APT540. APT540was tested for antimicrobial activity against E. coli strain TG1 and B.subtilis 168S as described in Methods. The minimum inhibitoryconcentration of APT540 to prevent the growth of E. coli strain TG1 was0.067 mg/mL after 6 h and 0.2 mg/mL after 16 h. The minimum inhibitoryconcentration of APT540 to prevent the growth of B. subtilis strain 168Swas 0.007 mg/mL after 6 h and 24 h growth.

Example 3 Synthesis and Characterization of APT541

[0195] APT541 is an N-myristoyl derivative of SEQ ID NO:1, which isfurther derivatised at its C-terminus by the addition of a cysteineresidue on the side chain of the C-terminal cysteine.

[0196] APT542 (Example 1; 10 mM in 100 mM Tris pH 8.5; 1.3 mL) was mixedwith 100 mM cysteine (0.1 mL) and stirred over 2 h. The reaction mixturewas purified by preparative HPLC using a gradient of 0-100 %acetonitrile in 0.1% trifluoroacetic acid over 10 minutes. The producteluted at approximately 10.0 minutes, and was collected. Evaporation ofvolatiles and lyophilisation afforded APT541 as a white solid. MALDI TOFMass Spec. C92H168N26O26S2 requires: 2117.2 Da. Found: 2117.3 Da. Whenpurified APT541 was treated with excess 1 mM DTT, no increase inabsorbance at 343 nm was observed which indicated that all thiopyridylgroups had been replaced.

[0197] The antibacterial activity of APT541 was also determined againstE. faecium STR 207, E. faecalis V 583, E. faecium STR 211, and S. aureusH, as described in Methods. The minimum inhibitory concentration ofAPT541 was 0.128 mg/mL, 0.256 mg/mL, 0.064 mg/mL, and 0.064 mg/mL foreach microorganism respectively.

Example 4 Synthesis and Characterisation of APT537

[0198] The peptide of SEQ ID NO:2 was synthesised using the same overallmethodology as used in Example 1. The most significant change was thereplacement of three of the lysine residues by aspartate residues. Thepeptide was linked at its N-terminus to a myristoyl group, and anS-linked 2-thiopyridyl group introduced as in example 1.

Example 5 Synthesis and Characterisation of APT539

[0199] APT539 was synthesised using the same overall methodology as usedin Example 1, using the peptide of SEQ ID NO:1. The most significantdifference was the replacement of myristic acid with lauric acid.

Example 6 Synthesis and Characterisation of APT538

[0200] APT538 was synthesised using the same overall methodology as usedin Example 1, using the peptide of SEQ ID NO:1. The most significantdifference was the replacement of myristic acid with decanoic acid.

Example 7 Synthesis and Characterisation of APT171

[0201] APT171 was synthesised using the same overall methodology as usedin Example 1, using the peptide of SEQ ID NO:1. The most significantdifference was the replacement of myristic acid with octanoic acid.

Example 8 Synthesis and Characterisation of APT170

[0202] APT170 was synthesised using the same overall methodology as usedin Example 1, using the peptide of SEQ ID NO:1. The most significantdifference was the replacement of myristic acid with butanoic acid.

Example 9 Synthesis and Characterisation of APT2197

[0203] APT2197 was synthesised using the same overall methodology asused in Example 1, using the peptide of SEQ ID NO:1. The mostsignificant difference was the replacement of myristic acid by palmiticacid.

Example 10 Synthesis and Characterisation of APT2198

[0204] APT2198 was synthesised using the same overall methodology asused in Example 1, using the peptide of SEQ ID NO:1. The mostsignificant difference was the replacement of myristic acid withoctadecanoic acid.

Example 11 Synthesis and Characterisation of APT2199

[0205] APT2199 was synthesised using the same overall methodology asused in Example 1, using the peptide of SEQ ID NO:1. The mostsignificant difference was the replacement of myristic acid witheicosanoic acid.

Example 12 Synthesis and Characterisation of APT2200

[0206] APT2200 was synthesised using the same overall methodology asused in Example 1, using the peptide of SEQ ID NO:1. The mostsignificant difference was the replacement of myristic acid with4-biphenyl-carboxylic acid.

Example 13 Synthesis and Characterisation of APT2235

[0207] APT2235 was synthesised using the same overall methodology asused in Example 1, using a peptide of SEQ ID NO:3, and omittingattachment of a group at the N-terminus, but retaining the 2-thiopyridylderivatisation at the C-terminus. The LC50 was determined to be 1.2 μM.

Example 14 Synthesis of APT2036N-(Myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 3)

[0208] APT2036, was synthesised chemically in three steps fromvancomycin. Vancomycin hydrochloride (100 mg, 0.0673 mmol) was dissolvedin dry dimethylformamide (1 mL) and dry methyl sulfoxide (1 mL).2-Aminoethyl-2′-pyridyldisulfide dihydrochloride (34.8 mg, 0.1346 mmol)was added and the mixture cooled to 0° C. under an atmosphere of drynitrogen. 2-(1-Hydroxybenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (0.234 mL of a 0.45 M solution in drydimethylformamide, 0.1 mmol) and hydroxybenzotriazole (1 mg, cat.) wereadded, the mixture allowed to warm to ambient temperature, and stirredfor 6 h. The reaction was characterised by the disappearance ofvancomycin and the appearance of the product (APT2033-Structure 1 asshown in the table below) by HPLC. The product was purified usingpreparative HPLC using a gradient of 10-90% buffer B in buffer A over 20minutes (buffer A: 0.1% trifluoroacetic acid; buffer B: 90%acetonitrile, 0.1% trifluoroacetic acid), and a Jupiter C18, 250×10 mm,300 Å column running at 5 mL/min. The volatile components were removedin vacuo and the aqueous solution lyophilised to afford APT2033 as awhite hydrochloride salt. Retention time 8.4 min; MALDI TOF Mass Spec.C73H86C13N11O22S2 requires: 1640.0 Da. Found. 1638.5 Da. Theantibacterial activity of APT2033 was determined against E. faecium STR207, E. faecalis V 583, E. faecium STR 211, and S. aureus H, asdescribed in Methods. The minimum inhibitory concentration of APT2033was 0.256 mg/mL, 0.032 mg/mL, 0.002 mg/mL, and 0.004 mg/mL for eachmicroorganism respectively. LC50 No lysis observed at highestconcentration of 166 μM.

[0209] APT2033 (9.9 mg, 0.00617 mmol) was dissolved in water (1 mL) andtris-2-carboxyethyl phosphine (1.9 mL of a 10 mM solution in 50 mM HEPESpH 4.5) added with stirring. The mixture was stirred over 30 minutes andthe product (APT2035 -Structure 2) purified and isolated as for APT2033. Retention time 7.0 min; MALDI TOF Mass Spec. C68H83Cl3N10O22Srequires: 1530.9 Da. Found. 1529.1 Da. The antibacterial activity ofAPT2035 was also determined against E. faecium STR 207, E. faecalis V583, E. faecium STR 211, and S. aureus H, as described in Methods. Theminimum inhibitory concentration of APT2035 was 0.128 mg/mL, 0.016mg/mL, 0.002 mg/mL, and 0.008 mg/mL for each microorganism respectively.LC50 No lysis observed at highest concentration of 166 μM.

[0210] APT2035 (1.62 mg, 0.00108 mmol) was dissolved in water (0.2 mL)and MSWP1 (0.04 mL of a 21.6 mM solution in dry methyl sulfoxide, 0.87μmol) added. The mixture was stirred over 2 h before the product(APT2036-Structure 3 as shown in the table below) was purified bypreparative HPLC (10-90 % buffer B over 20 minutes) and isolated as itshydrochloride salt as for APT2033. Retention time 13.5 min; MALDI TOFMass Spec. C157H244C13N35O46S2 requires: 3528.4 Da. Found. 3528.1 Da.The antibacterial activity of APT2036 was also determined against E.faecium STR 207, E. faecalis V 583, E. faecium STR 211, and S. aureus H,as described in Methods. The minimum inhibitory concentration of APT2036was 0.008 mg/ml, 0.008 mg/ml, 0.008 mg/ml, and 0.004 mg/ml for eachmicroorganism respectively. LC50 71 uM.

Example 15 Synthesis of APT2037N-(Lauroyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 4)

[0211] APT2037 was synthesised using the same overall methodology asused in Example 9. The most significant difference was the replacementof APT542 with APT539, to give the structure shown. Retention time 11.9min; The antibacterial activity of APT2037 was also determined againstE. faecium STR 207, E. faecalis V 583, E. faecium STR 211, and S. aureusH, as described in Methods. The minimum inhibitory concentration ofAPT2037 for each microorganism was 0.016 mg/ml, 0.016 mg/ml, 0.004mg/ml, and 0.008 mg/ml respectively. LC30 71 uM.

Example 16 Synthesis of APT2038N-(Decanoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 5)

[0212] APT2038 was synthesised using the same overall methodology asused in Example 9. The most significant difference was the replacementof APT542 with APT538, to give the structure shown. Retention time 10.7min; The antibacterial activity of APT2038 was also determined againstE. faecium STR 207, E. faecalis V 583, E. faecium STR 211, and S. aureusH, as described in Methods. The minimum inhibitory concentration ofAPT2038 for each microorganism was 0.064 mg/ml, 0.064 mg/ml, 0.002mg/ml, and 0.008 mg/ml respectively. LC10 71 uM.

Example 17 Synthesis of APT2039N-(Octanoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 6)

[0213] APT2039 was synthesised using the same overall methodology asused in Example 9. The most significant difference was the replacementof APT542 with APT171, to give the structure shown. Retention time 9.0min; The antibacterial activity of APT2039 was also determined againstE. faecium STR 207, E. faecalis V 583, E. faecium STR 211, and S. aureusH, as described in Methods. The minimum inhibitory concentration ofAPT2039 for each microorganism was 0.128 mg/ml, 0.128 mg/ml, 0.002mg/ml, and 0.0016 mg/ml respectively. LC30 no lysis at 71 uM.

Example 18 Synthesis of APT2040N-(n-Butyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 7)

[0214] APT2040 is synthesised using the same overall methodology as usedin Example 9. The most significant difference is the replacement ofAPT542 with APT170, to give the structure shown.

Example 19 Synthesis of APT2041N-(Myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Asp-Lys-Asp-Lys-Asp-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 8)

[0215] APT2041 was synthesised using the same overall methodology asused in Example 9. The most significant difference is the replacement ofAPT542 with APT537, to give the structure shown. Retention time 14.1min; The antibacterial activity of APT2041 was also determined againstE. faecium Str 207, E. faecalis V 583, E. faecium STR 211, and S. aureusH, as described in Methods. The minimum inhibitory concentration ofAPT2041 Was 0.016 mg/ml, 0.016 mg/ml, 0.004 mg/ml, and 0.008 mg/ml foreach microorganism respectively. LC50 4.5 uM.

Example 20 Synthesis of APT2208N-(Palmitoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 9)

[0216] APT2208 was synthesised using the same overall methodology asused in Example 9. The most significant difference is the replacement ofAPT542 with APT2197, to give the structure shown. Retention time 15.2min; The antibacterial activity of APT2208 was also determined againstE. faecium STR 207, E. faecalis V 583, E. faecium STR,211,. and S.aureus H, as described in Methods. The minimum inhibitory concentrationof APT2208 was 0.008 mg/ml, 0.016 mg/ml, 0.002 mg/ml, and 0.004 mg/mlfor each microorganism respectively.

Example 21 Synthesis of APT2209N-(Octadecanoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 10)

[0217] APT2209 was synthesised using the same overall methodology asused in Example 9. The most significant difference is the replacement ofAPT542 with APT2198, to give the structure shown. Retention time 16.7min; The antibacterial activity of APT2209 was also determined againstE. faecium STR 207, E. faecalis V 583, E. faecium STR 211, and S. aureusH, as described in Methods. The minimum inhibitory concentration ofAPT2209 was 0.032 mg/ml, 0.064 mg/ml, 0.016 mg/ml, and 0.032 mg/ml foreach microorganism respectively.

Example 22 Synthesis of APT2210N-(Eicosanoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 11)

[0218] APT2210 was synthesised using the same overall methodology asused in Example 9. The most significant difference is the replacement ofAPT542 with APT2199, to give the structure shown. Retention time 18.45min; The antibacterial activity of APT2210 was also determined againstE. faecium STR 207, E. faecalis V 583, E. faecium STR 211, and S. aureusH, as described in Methods. The minimum inhibitory concentration ofAPT2210 was 0.128 mg/ml, 0.128 mg/ml, 0.032 mg/ml, and >0.128 mg/ml foreach microorganism respectively.

Example 23 Synthesis of APT2211N-(4-Biphenylcarboxyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-thioethyl-2-vancomycincarboxamide)-Cys-NH2 (Structure 12) APT2211 was synthesised using thesame overall methodology as used in Example 9. The most significantdifference is the replacement of APT542 with APT2200, to give thestructure shown. Retention time 9.3 min; The antibacterial activity ofAPT2211 was also determined against E. faecium STR 207, E. faecalis V583, E. faecium STR 211, and S. aureus H, as described in Methods. Theminimum inhibitory concentration of APT2211 was 0.008 mg/ml, 0.032mg/ml, <0.03 mg/ml, and 0.00025 mg/ml for each microorganismrespectively. LC50 no lysis at 166 M. Example 24 Synthesis of APT2122N-(Myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-2-thioethyl-succinyl-vancomycinvancosaminide)-Cys-NH2 (Structure 15)

[0219] APT2122 was synthesised chemically in three steps fromvancomycin. Vancomycin hydrochloride (100 mg, 0.0673 mmol) was dissolvedin dry dimethylformamide (4 mL) and DIEA (12.9 μL, 0.0742 mmol) added.2-Aminoethyl-2′-pyridyldisulfide dihydrochloride (0.500 g, 1.93 mmol)was dissolved in water (20 mL) and dichloromethane (20 mL) added. 1 MNaOH was added dropwise to take the pH to 12, the organic layerextracted, dried over anhydrous magnesium sulphate, and filtered.Succinic anhydride (193 mg, 1.93 mmol) and DIEA (0.5 mL) were added andthe mixture stirred at ambient temperature over 1 h. 1.04 mL Of thismixture was evaporated and dissolved in DMF (300 μL). PyBOP (57.8 mg,111.1 μmol) And DIEA (19.3 μL, 111.1 μmol) were added, the mixturestirred over 15 mins, then added to the solution of vancomycin. After 6h the reaction was characterised by the disappearance of vancomycin andthe appearance of the product (APT2116-Structure 13) by HPLC. Theproduct was purified as for APT2036 to afford APT2116 (structure 9) as awhite hydrochloride salt. Retention time 8.2 min; MALDI TOF Mass Spec.C77H90Cl3N11O25S2 requires: 1740.1 Da. Found. 1739.4 Da. Theantibacterial activity of APT2116 was determined against E. faecium STR207, E. faecalis V 583, E. faecium STR 211, and S. aureus H, asdescribed in Methods. The minimum inhibitory concentration of APT2116was 0.512 mg/mL, 0.256 mg/mL, 0.032 mg/mL, and >0.512 mg/mL for eachmicroorganism respectively.

[0220] APT2116 (8.3 mg, 0.00477 mmol) was dissolved in water (0.5 mL)and tris-2-carboxyethyl phosphine (1.3 mL of a 10 mM solution in 50 mMHEPES pH 4.5) added with stirring. The mixture was stirred over 30minutes and the product (APT2117-Structure 14) purified and isolated asfor APT 2116. Retention time 7.1 min; MALDI TOF Mass Spec.C72H87C13N10O25S requires: 1630.9 Da. Found. 1632.0 Da. Theantibacterial activity of APT2117 was also determined against E. faeciumSTR 207, E. faecalis V 583, E. faecium STR 211, and S. aureus H, asdescribed in Methods. The minimum inhibitory concentration of APT2117was >512 mg/mL, 512 mg/mL, 0.008 mg/mL, and 0.008 mg/mL for eachmicroorganism respectively.

[0221] APT2117 (1.41 mg, 0.864 mmol) was dissolved in water (0.20 mL)and MSWP1 (0.022 mL of a 21.6 mM solution in dry methyl sulfoxide, 0.475μmol) added. The mixture was stirred over 2 h before the product(APT2122-Structure 15) was isolated as for APT2116. Retention time 13.8min; MALDI TOF Mass Spec. C157H243Cl2N35O46S2 requires: 3628.4 Da.Found. 3638.4 Da. The antibacterial activity of APT2122 was alsodetermined against E. faecium STR 207, E. faecalis V 583, E. faecium STR211, and S. aureus H, as described in Methods. The minimum inhibitoryconcentration of APT2122 was 0.008 mg/ml, 0.016 mg/ml, 0.008 mg/ml, and0.016 mg/ml for each microorganism respectively.

Example 25 Synthesis of APT2237Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-[(S-2-thioethyl-succinyl-vancomycinvancosaminide)-Cys]—NH2 (Structure 16)

[0222] APT2237 was synthesised using the same overall methodology asused in Example 24. The most significant difference is the replacementof APT542 with APT2235, to give the structure shown. Retention time 14.1min; The antibacterial activity of APT2237 was also determined againstE. faecium STR 207, E. faecalis V 583, E. faecium STR 211, and S. aureusH, as described in Methods. The minimum inhibitory concentration ofAPT2237 was 0.016 mg/ml, 0.064 mg/ml, 0.016 mg/ml, and 0.008 mg/ml foreach microorganism respectively.

Summary of Biological Results

[0223] The antibacterial activities measured above are summarised in thefollowing table, where all entries represent the maximum inhibitorycomposition, expressed in mg/ml. E. faecium E. faecalis E. faecium STR207 V583 STR 211 S. aureus H APT 542 0.064 0.256 0.032 0.064 APT 5410.128 0.256 0.064 0.064 APT 2033 0.256 0.032 0.002 0.004 APT 2035 0.1280.016 0.002 0.008 APT 2036 0.008 0.008 0.008 0.004 APT 2037 0.016 0.0160.004 0.008 APT 2038 0.064 0.064 0.002 0.008 APT 2039 0.128 0.128 0.0020.0016 APT 2116 0.512 0.256 0.032 >0.512 APT 2117 >0.512 0.512 0.0080.008 APT 2122 0.008 0.016 0.008 0.016 APT 2208 0.008 0.016 0.002 0.004APT 2041 0.016 0.016 0.004 0.008 APT 2209 0.032 0.064 0.016 0.032 APT2210 0.128 0.128 0.032 >0.128 APT 2211 0.008 0.032 <0.00003 0.00025 APT2212 0.008 0.016 0.008 0.016 APT 2237 0.016 0.064 0.016 0.008

[0224] TABLE OF CHEMICAL STRUCTURES Structures 1-12

Structure 1: APT 2033 R = 2-thiopyridyl Structure 2: APT 2035 R = HStructure 3: APT 2036 R = Myristoyl-SEQ ID NO: 1-NH2 Structure 4: APT2037 R = Lauroyl-SEQ ID NO: 1-NH2 Structure 5: APT 2038 R = Decanoyl-SEQID NO: 1-NH2 Structure 6: APT 2039 R = Octanoyl-SEQ ID NO: 1-NH2Structure 7: APT 2040 R = n-Butyl-SEQ ID NO: 1-NH2 Structure 8: APT 2041R = Myristoyl-SEQ ID NO: 2-NH2 Structure 9: APT 2208 R = Palmitoyl-SEQID NO: 1-NH2 Structure 10: APT 2209 R = Octadecanoyl-SEQ ID NO: 1-NH2Structure 11: APT 2210 R = Eicosanoyl-SEQ ID NO: 1-NH2 Structure 12: APT2211 R = N-(4-Biphenylcarboxyl)-SEQ ID N0: 1-NH2 Structures 13-16:

Structure 13: APT 2116 R = 2-thiopyridyl Structure 14: APT 2117 R = HStructure 15: APT 2122 R = Myristoyl-SEQ ID NO: 1-NH2 Structure 16: APT2237 R = SEQ ID NO: 3-NH2

[0225] SEQUENCE LISTING GSSKSPSKKKKKKPGDC SEQ ID NO:1 GSSKSPSKDKDKDPGDCSEQ ID NO:2 GIGAVLKVLTTGLPALISWIKRKRQQC SEQ ID NO:3 DGPKKKKKKSPSKSSG SEQID NO:4 GSSKSPSKKKKKKPGD SEQ ID NO:5 SPSNETPKKKKKRFSFKKSG SEQ ID NO:6DGPKKKKKKSPSKSSK SEQ ID NO:7 SKDGKKKKKKSKTK SEQ ID NO:8GIGKFLHSAGKFGKAFVGEIMK SEQ ID NO:9 GIGKFLHSAKKFGKAFVGEIMNS SEQ ID NO:10VGALAVVVWLWLWLW SEQ ID NO:11 GLLSVLGSVAKHVLPHVVPVIAEHL SEQ ID NO:12FLGGLIKIVPAMICAVTKKC SEQ ID NO:13 GLFGVLAKVAAHVVPAIAEHF SEQ ID NO:14VKLKVYPLKVKLYP SEQ ID NO:15 ILPWKWPWWPWRR SEQ ID NO:16 cyclizedisooctanoyl BTBB (BFdLBBT) SEQ ID NO:17 KWKSFIKKLTSAAKKVVTTAKPLISS SEQID NO:18 KWKLFKKIGIGAVLKVLTTGLPALTLTK SEQ ID NO:19KWKSFIKKLTTAVKKVLTTGLPALIS SEQ ID NO:20 ILKKWPWWPWRRK-NH2 SEQ ID NO:21KWKLFKKIGIGAVLKVLTTGLPALIS SEQ ID NO:22 Cyclized RL (CRIVVIRVC)R SEQ IDNO:23 RLCRIVVIRVCR SEQ ID NO:24 RLSRIVVIRVSR SEQ ID NO:25 cyclic(PFdLOVPFdLoV) SEQ ID NO:26 cyclic (PVKLKVdYdPLKVKLYd) SEQ ID NO:27ILPWKWPWWPWRR-NH2 SEQ ID NO:28 GIGAVLKVLTTGLPALISWIKRKRQQ SEQ ID NO:29GIGAdVLKdVLTTGLPALdISWIdKRKRQQ SEQ ID NO:30 GIGKFLKKAKKFGKAFVKILKK SEQID NO:31 GIGKFLHSAKKFGKAFVAEIMNS SEQ ID NO:32 GIAKFLHSAKKFGKAFVAEIMNSSEQ ID NO:33 AAGKFLHSAKKFGKAFVGDIMNS SEQ ID NO:34G-GKFLHSAKKFGKAFVGEIMNS SEQ ID NO:35 G-GKFIHSAKKFGKAFVGEIMNS SEQ IDNO:36 GIGKFIHSAKKFGKAFVGEIMNSK SEQ ID NO:37 GIGAVLKVLTTGLPALISWIKRKRQQCSEQ ID NO:38 L-Alanine-γ-D-Glutamate-Xaa-D- SEQ ID NO:39Alanine-D-Alanine

1. An antibacterial compound of formula V-L-W—X wherein V is aglycopeptide moiety which inhibits peptidoglycan biosynthesis inbacteria; L is a linking group; W is a peptidic membrane-associatingelement; and X is hydrogen or a membrane-insertive element.
 2. Acompound according to claim 1 wherein V-L- is of the formula:

or salt thereof, in which: Y and Y′ are independently hydrogen orchloro; R is hydrogen, 4-epi-vancosaminyl, actinosaminyl, ristosaminyl,or a group of the formula —R^(a)-L- wherein Ra is 4-epi-vancosaminyl,actinosaminyl, ristosaminyl and L is attached to the amino group ofR^(a); R¹ is hydrogen, or mannose; R² is —NH₂, —NHCH₃, —N(CH₃)₂, —NHL-,or —N(CH₃)L- R³ is —CH₂CH(CH₃)₂, [p-OH, m-Cl]phenyl, p-rhamnose-phenyl,[p-rhamnose-galactose]phenyl, [p-galactose-galactose]phenyl, or[p-CH₃O-rhamnose]phenyl; R⁴ is —CH₂—(CO)NH₂, benzyl, [p-OH]phenyl, or[p-OH, m-Cl]phenyl; R⁵ is hydrogen, or mannose, R⁶ is hydrogen,4-epi-vancosaminyl, vancosaminyl, actinosaminyl, ristosaminyl, oracosaminyl; or R⁶ is a group of the formula R^(b)-L- wherein R^(b) is4-epi-vancosaminyl, vancosaminyl, actinosaminyl, ristosaminyl oracosaminyl and L is attached to the amino group of R^(b); or R⁶ is agroup R^(b)-R⁷ wherein R⁷ is an organic side chain moiety which is nomore than 1000 Da; Ter1 is hydroxy or -L-; provided that the compoundincludes at least one group -L-.
 3. A compound according to claim 2wherein the organic side chain moiety include those of the formula—CH₂—R⁸, in which R⁸ is hydrogen, alkyl of C₁-C₁₅, alkenyl of C₂-C₁₅,alkynyl of C₂-C₁₅, haloalkyl of C₁-C₇, acenaphthenyl, 2-fluorenyl,9,10-dihydro-2-phenanthrenyl, R⁹, alkyl of C₁-C₁₁—R⁹, alkenyl ofC₂-C₇—R⁹, alkynyl of C₂-C₇—R^(9,) or alkyl of C₁-C₇—O—R⁹ wherein R⁹ is aradical of the formula: —R¹⁰-[linker_((0 or 1))—R¹⁰]_((0 or 1)) whereineach R¹⁰ independently represents phenyl, cycloalkyl of C₅-C₆, naphthyl,or thienyl, each of which is unsubstituted or is optionally substitutedwith one or two substituents, each of which is independently alkyl ofC₁-C₁₀, haloalkyl of C₁-C₂, haloalkoxy of C₁-C₂, alkoxy of C₁-C₁₀, halo,cyano, or nitro; and “linker” is -alkylene of C₁-C₃, —O-alkylene ofC₁-C₆, -alkylene of C₁-C₆—O—, —O—, —N(H or lower alkyl of C₁-C₃)—, —S—,—SO—, —SO₂—, —NH—C(O)—, —C(O)—NH—, —CH═CH—, —C≡C—, —N═N—, —O—C(O)—, or—C(O)—O—.
 4. A compound according to any one of the preceding claimswherein V is selected from vancomycin, chloroeremomycin, teicoplanin,A₂-2, ristocetin A, eremomycin, balkimycin, actinodin A, complestanin,chloropeptin 1, kistamycin A and avoparcin.
 5. A compound according toany one of the preceding claims wherein W comprises a membrane bindingpeptide including at least one sequence (Xaa)_(n), where n is from 1 to12 and each Xaa is independently lysine or arginine.
 6. A compoundaccording to any one of the preceding claims wherein, W comprises amembrane-binding peptide comprising from 2 to 10 contiguous residuesselected from lysine and arginine.
 7. A compound according to claim 6wherein the membrane-binding peptide comprises from 7 to 30 amino acids.8. A compound according to claims 6 or 7 wherein the membrane-bindingpeptide is selected from the group: DGPKKKKKKSPSKSSG (SEQ ID NO: 4)GSSKSPSKKKKKKPGD (SEQ ID NO: 5); SPSNETPKKKKKRFSFKKSG (SEQ ID NO: 6);DGPKKKKKKSPSKSSK (SEQ ID NO: 7); and SKDGKKKKKKSKTK (SEQ ID NO: 8).
 9. Acompound according to any, one of the preceding claims wherein Wcomprises a membrane-inserting peptide.
 10. A compound according toclaim 9 wherein said membrane-inserting peptide is selected fromα-defensins; β-defensins; and bacteriocins.
 11. A compound according toclaim 10 wherein said β-defensin is a tachyplesin.
 12. A compoundaccording to claim 9 wherein said membrane-inserting peptide is amagainin.
 13. A compound according to claim 9 wherein saidmembrane-inserting peptide is maculatin 1.1 or caerin 1.1.
 14. Acompound according to any one of the preceding claims wherein X is alipophilic chain which has the following parameters: having from 6 to 30carbon atoms including those of any aromatic rings, if present; beingstraight or branched, and in the case of the latter containing one tothree branch points; being saturated or unsaturated, in the case of thelatter containing one to four double or triple bonds; optionally having1, 2 or 3 heteroatoms (in addition to those, if present, in aromaticrings, if present), independently selected from S, O or N; optionallycontaining one or more, for example two or three, aromatic rings, whichmay be fused and each of which may contain from 1, 2 or 3 heteroatomswhich, if present, are independently selected from N, O or S; andoptionally having from one to three substituents selected from hydroxy,—SH, amino and halo.
 15. A compound according to any one of claims 1 to14 for use in a method of treatment or prophylaxis of the human oranimal body.
 16. Use of compound according to any one of claims 1 to 14for the manufacture of a medicament for the treatment or prophylaxis ofa bacterial infection.
 17. A method of treating a bacterial infection ina subject which method comprises administering to said subject aneffective amount of the antibacterial agent of any one of claims 1 to14.
 18. A composition comprising a compound according to any one ofclaims 1 to 14 and a pharmaceutically acceptable carrier.